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Introduction: As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1 month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy., Methods: Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was measured by flow cytometry., Results: Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum antibody present at one month following vaccination., Conclusions: RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine, consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that the increased incidence and severity of infection observed in this patient population could be a consequence of diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination and from vaccines with enhanced immunogenicity.

Objective: Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes., Methods: (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay., Results: After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production., Conclusion: The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

Systemic lupus erythematosus (SLE) is a complex disease characterized by numerous autoantibodies and clinical involvement in multiple organ systems. The immunologic events triggering the onset and progression of clinical manifestations have not yet been fully defined, but a central role for B cells in the pathogenesis has been brought to the fore in the last several years. The breakdown of B-cell tolerance is likely a defining and early event in the disease process and may occur by multiple pathways, including alterations in factors that affect B-cell activation thresholds, B-cell longevity, and apoptotic cell processing. Antibody-dependent and -independent mechanisms of B cells are important in SLE. Thus, autoantibodies contribute to autoimmunity by multiple mechanisms including immune complex mediated type III hypersensitivity reactions, type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines including interferon alpha, tumor necrosis factor, and interleukin 1. Recent data have highlighted the critical role of toll-like receptors as a link between the innate and adaptive immune system in SLE immunopathogenesis. Given the large body of evidence implicating abnormalities in the B-cell compartment in SLE, there has been a therapeutic focus on developing interventions that target the B-cell compartment. Several different approaches to targeting B cells have been used, including B-cell depletion with monoclonal antibodies against B-cell-specific molecules, induction of negative signaling in B cells, and blocking B-cell survival and activation factors. Overall, therapies targeting B cells are beginning to show promise in the treatment of SLE and continue to elucidate the diverse roles of B cells in this complex disease., (Copyright 2010 Elsevier Inc. All rights reserved.)

B cells are critical players in the orchestration of properly regulated immune responses, providing protection against infectious agents without inflicting autoinflammatory damage. A balanced B cell compartment is also essential to create protective immunity in response to vaccines. This difficult compromise is achieved through the finely regulated participation of multiple B cell populations with different antibody-dependent and independent functions. Both types of functions allow B cells to powerfully modulate other components of the innate and adaptive immune system. For the most part, however, the necessary division of labor among different B cell populations is poorly understood. B cell dysfunction has been implicated in multiple autoimmune conditions. The physiological importance and complexity of B cell functions has been brought to the fore in recent years by the success of rituximab-based B cell depletion therapy (BCDT) in multiple autoimmune diseases including rheumatoid arthritis (RA) and multiple sclerosis (MS) which are conventionally viewed as T-cell mediated conditions. Given the widespread utilization of BCDT in malignant and autoimmune diseases and the key role of B cells in both protective immunity and pathogenic autoimmunity, a better understanding of B cell functions is of the essence and a focus of the research in our division. We are investigating these issues through a variety of approaches, including the study of the phenotype and function of human B cell populations in health, their perturbation in autoimmune disease states, the effects of targeted biologic therapies, and the study of relevant murine models.

Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.

Recently, two large randomized controlled trials of distinct biologic therapies in systemic lupus erythematosus, B-cell depletion with rituximab and co-stimulatory blockade with CTLA4Ig (abatacept), failed to meet primary endpoints. Given the great need for new treatments in lupus, these results were met with disappointment and have left the rheumatology and immunology community searching for an explanation. Are these experimental agents ineffective in lupus or are there trial design issues or other considerations? In this commentary, we discuss our perspective on these results within the context of current understanding of the pathophysiology of lupus and the mechanism of action of biologic therapies.

The introduction of biological agents in the treatment of systemic vasculitis offers the promise of targeted therapy with greater efficacy and fewer side effects than conventional treatments. In this paper, we review the rationale for biological strategies in vasculitis and discuss the results of clinical studies to date. The biotherapies discussed include immune-cell-depleting agents, both B- and T-cell targeted; costimulatory blockade; and cytokine blockade. Although most of these agents remain unproven until ongoing randomized clinical trials are complete, their introduction heralds a new era of vasculitis treatment and has provided novel insights into disease pathogenesis.

Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system characterized by severe episodes of optic nerve and spinal cord inflammation. NMO-IgG (anti-aquaporin-4) has been recently described as a sensitive and specific marker for NMO. As there have been prior published reports of an association between NMO and systemic autoimmune diseases, the prognostic value of the antibody test in these cases is uncertain. We describe a 47-year old woman with recurrent transverse myelitis and a long-standing history of systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome (APLS). While she did not have a history of optic neuritis, serological testing for the NMO-IgG was positive when she was admitted for her second episode of transverse myelitis. Testing for the NMO-IgG in cases of isolated or recurrent transverse myelitis attributed to current SLE and APLS may help clarify the diagnosis of a distinct disease process likely to cause recurrent and severe disability, warranting more aggressive immunotherapy.

Rheumatoid arthritis (RA) is mediated by a proinflammatory cytokine network with TNF at its apex. Accordingly, drugs that block TNF have demonstrated significant efficacy in the treatment of RA. A great deal of experimental evidence also strongly implicates B cells in the pathogenesis of RA. Yet, it remains unclear whether these two important players and the therapies that target them are mechanistically linked. In this study we demonstrate that RA patients on anti-TNF (etanercept) display a paucity of follicular dendritic cell networks and germinal center (GC) structures accompanied by a reduction in CD38+ GC B cells and peripheral blood memory B cell lymphopenia compared with healthy controls and RA patients on methotrexate. This study provides initial evidence in humans to support the notion that anti-TNF treatment disrupts GC reactions at least in part via effects on follicular dendritic cells.

Objective: Recent data suggest that the reconstituting peripheral B cell compartment after B cell depletion therapy may be functionally immature, with a preponderance of transitional B cells and a paucity of memory B cells. This study was undertaken to determine the magnitude, duration, and cause of these defects in rituximab-treated systemic lupus erythematosus (SLE) patients., Methods: Fifteen patients with SLE previously treated with rituximab as part of a phase I/II dose-escalation study were evaluated during a long-term followup (mean followup period 41 months). B cells from peripheral blood and tonsils were assessed using multicolor flow cytometry, and their developmental pathway was classified based on the expression of defined surface markers., Results: Reconstitution of peripheral blood CD27+ memory B cells was delayed for several years after B cell depletion therapy in a subset of patients with prolonged clinical responses and autoantibody normalization. This delay correlated with the degree of expansion of B cells of a transitional phenotype during the B cell reconstitution phase (P = 0.005) and the absence of baseline autoantibodies directed against extractable nuclear antigens (RNP, Sm, Ro antigen, La antigen). Despite the paucity of peripheral blood memory cells and the prolonged expansion of functionally immature transitional B cells, tonsil biopsy tissues revealed active germinal center (GC) reactions, but with decreased Fc receptor homolog 4-positive memory B cells., Conclusion: These results suggest heterogeneity in the B cell depletion and reconstitution process that impacts clinical and immunologic outcomes in SLE. The presence of GC reactions, but with altered memory B cell subpopulations in tonsils, suggests that peripheral blood memory cell reconstitution lags behind a slow secondary lymphoid tissue recovery, with important implications for immunologic competence and tolerance.

The long-term immunologic effects of B cell depletion with rituximab and the characteristics of the reconstituting B cell pool in lymphoma patients are not well defined, despite the widespread usage of this therapy. Here we report that during the B cell reconstitution phase a majority of the peripheral blood B cells have an immature transitional phenotype (47.8%+/-25.2% vs. 4.4%+/-2.4% for normal controls, p<0.0001), similar to what has been described during the original ontogeny of the immune system and following bone marrow transplantation. Moreover, the recovery of the CD27+ memory B cell pool was delayed compared to normal B cell ontogeny, remaining below normal controls at 1 year post-rituximab (4.4%+/-3% vs. 31%+/-7%, p<0.0001). Expansion of functionally immature B cells and decreased memory B cells may contribute to an immunodeficient state in patients recovering from rituximab mediated B cell depletion, particularly with repeated treatment.

Systemic lupus erythematosus (SLE) is a complex disease characterized by the production of autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset of clinical manifestations have not yet been fully defined, but a central role for B cells in the pathogenesis of this disease has been brought to the fore in the last several years by work performed at multiple laboratories in both mice and humans. B cell defects that have been defined include abnormal expression or function of key signaling molecules, dysregulation of cytokines with key B cell effects, and perturbations in B cell developmental subsets. Many of these defects may contribute to or be reflective of abnormalities in B cell tolerance. Both antibodydependent and antibody-independent mechanisms of B cells are important in SLE. Thus, autoantibodies contribute to autoimmunity by multiple mechanisms, including immunecomplex mediated type III hypersensitivity reactions and type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines, including IFNalpha, TNF, and IL-1. Autoantibody-independent B cell functions have been postulated to include antigen-presentation, T-cell activation and polarization, and dendritic cell (DC) modulation. Several of these functions are mediated by the ability of B cells to produce immuno-regulatory cytokines, chemokines, and lymphangiogenic growth factors and by their critical contribution to lymphoid tissue development and organization, including the development of ectopic tertiary lymphoid tissue. Given the large body of evidence implicating abnormalities in the B cell compartment in SLE,there has been a recent therapeutic focus on developing interventions that target the B cell compartment by multiple mechanisms.

Our understanding of the multiple physiological and pathological functions of B-cells continues to expand at a fascinating rate. A critical part of this expanding knowledge is the realization that B-cells can be responsible, at least in part, for diseases in which they had not been previously suspected and that their pathogenic influence can be mediated by multiple mechanisms. In turn, the availability of effective agents capable of inducing profound and long-lasting B-cell depletion and the safety and efficacy of Rituximab in non-Hodgkin lymphoma has prompted investigators to use this therapeutic approach in a large number of autoimmune diseases. Thus far, the results have been very promising, and in some cases nothing short of spectacular. In this review, we shall discuss the roles of B-cells in health and disease and the available evidence regarding the efficacy of B-cell depletion in human autoimmunity. Finally, we will discuss some of the many challenges and opportunities that the medical and scientific community should address in the foreseeable future.

Systemic lupus erythematosus (SLE) is a complex disease characterised by numerous autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset of clinical manifestations have not yet been fully defined, but a central role for B cells in the pathogenesis of this disease has more recently gained prominence as a result of research in both mice and humans. Both antibody-dependent and -independent mechanisms of B cells are important in SLE. Autoantibodies contribute to autoimmunity by multiple mechanisms, including immune complex-mediated type III hypersensitivity reactions, type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines such as interferon-alpha, tumour necrosis factor and interleukin-1. Suggested autoantibody-independent B-cell functions include antigen presentation, T-cell activation and polarisation, and dendritic-cell modulation. Several of these functions are mediated by the ability of B cells to produce immunoregulatory cytokines, chemokines and lymphangiogenic growth factors, and by their critical contribution to lymphoid tissue development and organisation, including the development of ectopic tertiary lymphoid tissue. Given the large body of evidence implicating abnormalities in the B-cell compartment in SLE, a recent therapeutic focus has been to develop interventions that target the B-cell compartment by multiple mechanisms.Rituximab, a mouse-human chimeric monoclonal antibody against CD20 that specifically depletes B cells, has been studied the most extensively. Although promising open-label data await confirmation in ongoing multicentre placebo-controlled trials, a number of preliminary conclusions can be drawn. The adequacy of peripheral B-cell depletion depends on achieving high and sustained serum rituximab concentrations, pharmacokinetics that can be varied with treatment dose and factors that may affect drug clearance, such as human anti-chimeric antibodies. In SLE patients with effective B-cell depletion, the clinical response can be significant, with favourable responses observed in a diverse array of disease manifestations. Moreover, rituximab appears to have the potential to induce clinical remission in severe, refractory disease. B-cell depletion has the potential to induce disease amelioration by inhibiting autoantibody production and/or by interfering with other B-cell pathogenic functions. The fact that clinical improvement correlates with B-cell depletion and precedes by several months any decline in serum levels of relevant autoantibodies suggests a predominant effect of autoantibody-independent functions of B cells, although the subset of patients with disease remission ultimately also experience autoantibody normalisation. Significant questions remain about rituximab therapy in SLE, including the immunological determinants of treatment response and remission, the role of combination therapy, and the safety of repeated courses of rituximab. In addition, the efficacy and role of other B-cell-depleting approaches, such as humanised anti-CD20 antibodies and anti-CD22, remain to be defined. Another B-cell-targeted therapeutic approach is to block costimulatory interactions between T and B cells. Blockade of the CD40-CD40 ligand pathway has met with variable clinical benefit and unfortunate thromboembolic complications, although inhibition of the B7 pathway with cytotoxic T-lymphocyte antigen-4Ig is currently under early investigation in SLE clinical trials. Preliminary data on the treatment of SLE with belimumab, a fully human monoclonal antibody that specifically binds to and neutralises the B-lymphocyte stimulator (BLyS or B-cell-activating factor [BAFF]), are now available. In a phase II double-blind, placebo-controlled trial of the safety and efficacy of three different doses administered in addition to standard therapy, belimumab was well tolerated but reportedly did not meet primary efficacy endpoints. Blockade of BAFF is still viewed as a promising therapeutic approach and additional agents that interfere with the BAFF pathway are under study.Overall, therapies targeting B cells appear to be promising in the treatment of SLE, provide additional evidence for the importance of B cells to disease pathogenesis, and will continue to elucidate the diverse roles of B cells in this disease.

Breach of B cell tolerance is central to the pathogenesis of systemic lupus erythematosus (SLE). However, how B cell tolerance is subverted in human SLE is poorly understood due to difficulties in identifying relevant autoreactive B cells and in obtaining lymphoid tissue. We have circumvented these limitations by using tonsil biopsies to study autoreactive B cells (9G4 B cells), whose regulation is abnormal in SLE. Here we show that 9G4 B cells are physiologically excluded during the early stages of the GC reaction before acquiring a centroblast phenotype. Furthermore, we provide evidence to indicate that an anergic response to B cell receptor stimulation may be responsible for such behavior. In contrast, in SLE, 9G4 B cells progressed unimpeded through this checkpoint, successfully participated in GC reactions, and expanded within the post-GC IgG memory and plasma cell compartments. The faulty regulation of 9G4 B cells was not shared by RA patients. To our knowledge, this work represents the first comparative analysis of the fate of a specific autoreactive human B cell population. The results identify a defective tolerance checkpoint that appears to be specific for human SLE.

Although systemic lupus erythematosus (SLE) is indeed a complex autoimmune disease, recent advances in our understanding of lupus pathogenesis have suggested new, targeted approaches to therapy. The purpose of this review is to discuss the underlying scientific rationale and results of first clinical studies of new treatment approaches to SLE, with a focus on cell-depleting therapies and cytokine blockade. It has become clear that the B lymphocyte plays a key role in disease pathogenesis by both autoantibody-dependent and autoantibody-independent mechanisms. Additionally, aberrant interactions between B and T cells are critical to disease emergence and progression. New agents that directly target immune cells abnormal in SLE include the B-cell depleting or modulating antibodies, rituximab (anti-CD20) and epratuzumab (anti-CD22) and the anti-dsDNA tolerogen LJP394. Another promising approach has been to block co-stimulatory interactions between T and B cells, for example by inhibiting the CD40-CD40 ligand pathway with anti-CD40 ligand monoclonal antibody or the B7 pathway with CTLA-4Ig. Immune cells can also be manipulated indirectly through cytokine effects. For B cells, anti-BAFF (B-cell activation factor of the tumor necrosis family) provides an example of this approach. Other, more pleiotropic cytokines can likewise be blocked in SLE. In addition to the blockade of interleukin-10 (IL-10), the first anti-cytokine approach examined, it is mainly anti-tumor necrosis factor therapy that has come into focus, holding promise for some patients with lupus nephritis. The majority of the available data on these new treatment approaches stems from open-label trials, but controlled trials are under way. Moreover, many additional cytokines, such as interleukin (IL)-6, IL-18, and the type I interferons, represent interesting future targets.

Objective: B lymphocyte depletion has recently emerged as a promising approach to the treatment of systemic lupus erythematosus (SLE). As part of a phase I/II dose-ranging trial of rituximab in the treatment of SLE, we evaluated the fate of discrete B cell subsets in the setting of selective depletion by anti-CD20 monoclonal antibody and during the B cell recovery phase., Methods: B cell depletion and phenotype were examined by flow cytometry of peripheral blood mononuclear cells for CD19, CD20, CD27, IgD, and CD38 expression. Changes in autoreactive B lymphocytes and plasma cells were assessed by determination of serum autoantibody levels (anti-double-stranded DNA and VH4.34) and by direct monitoring of a unique autoreactive B cell population bearing surface antibodies whose heavy chain is encoded by the VH4.34 gene segment., Results: Compared with normal controls, SLE patients displayed several abnormalities in peripheral B cell homeostasis at baseline, including naive lymphopenia, expansion of a CD27-,IgD- (double negative) population, and expansion of circulating plasmablasts. Remarkably, these abnormalities resolved after effective B cell depletion with rituximab and immune reconstitution. The frequency of autoreactive VH4.34 memory B cells also decreased 1 year posttreatment, despite the presence of low levels of residual memory B cells at the point of maximal B cell depletion and persistently elevated serum autoantibody titers in most patients., Conclusion: This study is the first to show evidence that in SLE, specific B cell depletion therapy with rituximab dramatically improves abnormalities in B cell homeostasis and tolerance that are characteristic of this disease. The persistence of elevated autoantibody titers may reflect the presence of low levels of residual autoreactive memory B cells and/or long-lived autoreactive plasma cells.

Anti-lymphocyte autoantibodies are a well-recognized component of the autoimmune repertoire in human systemic lupus erythematosus (SLE) and have been postulated to have pathogenic consequences. Early studies indicated that IgM anti-lymphocyte autoantibodies mainly recognized T cells and identified CD45, a protein tyrosine phosphatase of central significance in the modulation of lymphocyte function, as the main antigenic target on T cells. However, more recent work indicates that lupus autoantibodies can also recognize B cells and that CD45 may also represent their antigenic target. In particular, IgM Abs encoded by V(H)4.34 appear to have special tropism for B cells, and strong, but indirect evidence suggests that they may recognize a B cell-specific CD45 isoform. Because V(H)4.34 Abs are greatly expanded in SLE, in the present study we investigated the antigenic reactivity of lupus sera V(H)4.34 IgG Abs and addressed their contribution to the anti-lymphocyte autoantibody repertoire in this disease. Our biochemical studies conclusively demonstrate that lupus IgG V(H)4.34 Abs target a developmentally regulated B220-specific glycoform of CD45, and more specifically, an N-linked N-acetyllactosamine determinant preferentially expressed on naive B cells that is sterically masked by sialic acid on B220-positive memory B cells. Strikingly, our data also indicate that this reactivity in SLE sera is restricted to V(H)4.34 Abs and can be eliminated by depleting these Abs. Overall, our data indicate that V(H)4.34 Abs represent a major component of the lupus IgG autoantibody repertoire and suggest that the carbohydrate moiety they recognize may act as a selecting Ag in SLE.

Background: Posttransfusion purpura (PTP) is characterized by severe thrombocytopenia following blood transfusion that results from alloimmunization to platelet-specific alloantigens. Most cases involve antibodies against HPA-1a in homozygous HPA-1b persons., Case Report: A patient developed PTP after cardiopulmonary bypass associated with a platelet-specific antibody with strong reactivity against HPA-5a (Br(b)). Geno-typing confirmed that the patient was homozygous for HPA-5b., Conclusion: This is the first well-documented occurrence of PTP associated with isolated allosensitization to HPA-5a or Br(b). The case highlights the importance of maintaining a high level of suspicion for PTP in the appropriate clinical setting, even in an atypical patient.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by sun sensitivity, defective DNA repair, markedly increased susceptibility to skin cancer, and a variety of immunological defects, including defective natural killer (NK) cell activity. Retinoid therapy has been demonstrated to protect effectively against the development of skin cancers in patients with XP, although its mechanism of action is unknown. We describe a series of eight XP patients, six of whom were given oral isotretinoin. The NK cell activity was not affected by low-dose isotretinoin, i.e. 0.5 mg/kg per day. However, higher doses of isotretinoin, e.g. 1.0 mg/kg per day, produced a significant decrease in NK cell function, at the same time as producing a reduction in the frequency of development of skin cancers. Retinoid therapy may have a skin cancer preventing effect by enhancing other immune effector mechanisms or via epithelial cell differentiation.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E2-ER dimer per ERE. In contrast, highly purified E2-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E2-ER was 0.5 E2-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E2, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.

It has been suggested that cooperative binding of estrogen receptor (ER) may, in part, be responsible for the synergistic activation of transcription of estrogen-responsive genes that contain multiple estrogen-response elements (EREs). Experiments described here show that estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally occurring flanking sequences, such as an AT-rich region. In contrast, EREs lacking these sequences do not bind E2-ER cooperatively, regardless of ERE spacing or stereoalignment. Moreover, binding is of lower affinity and capacity in the absence of these critical flanking sequences. By varying the sequence of nucleotides adjacent to the ERE, features important for the flanking sequence effect were characterized. Interestingly, when ER was liganded with 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen, the antiestrogen-liganded ER complex (4-OHT-ER) did not bind cooperatively to multiple EREs, regardless of spacing or flanking sequence. We postulate that ERE flanking sequences bestow upon E2-ER enhanced ERE binding capacity and cooperativity, but do not affect 4-OHT-ER-ERE binding.

The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5'-GGTCAgagTGACC-3') but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E2-ER binding to AT-rich EREs, not cooperative. The affinity of E2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E2-ER binding to closely spaced EREs is more stable (Kd = 0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing (Kd = 2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E2-ER binding and may be critical for cooperative binding to stereoaligned EREs.